Oxidation-reduction sensitive green fluorescent protein variants

ABSTRACT

The disclosure provides proteins that can be used to determine the redox status of an environment (such as the environment within a cell or subcellular compartment). These proteins are green fluorescent protein (GFP) variants (also referred to as redox sensitive GFP (rosGFP) mutants), which have been engineered to have two cysteine amino acids near the chromophore and within disulfide bonding distance of each other. Also provided are nucleic acid molecules that encode rosGFPs, vectors containing such encoding molecules, and cells transformed therewith. The disclosure further provides methods of using the rosGFPs (and encoding molecules) to analyze the redox status of an environment, such as a cell, or a subcellular compartment within a cell. In certain embodiments, both redox status and pH are analyzed concurrently.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 10/471,857,filed Mar. 8, 2004, which is the § 371 U.S. National Stage ofInternational Application No. PCT/US02/07374, filed Mar. 11, 2002(published in English under PCT Article 21(2)), which in turn claims thebenefit of U.S. Provisional Patent Application Nos. 60/275,200, filedMar. 12, 2001, 60/293,427, filed May 23, 2001, and 60/302,894, filedJul. 3, 2001. These applications are incorporated herein in theirentirety.

STATEMENT REGARDING GOVERNMENT FUNDING

This invention was made with government support under grant numberGM07759-22 and grant number GM42618-10 both awarded by the NationalInstitutes of Health (NIH). The government may have certain rights inthe invention.

FIELD

The present disclosure relates to the field of genetic engineering, andin particular to green fluorescent protein (GFP) mutants that can beused to detect oxidation-reduction state, or a change inoxidation-reduction state.

BACKGROUND

The green fluorescent protein (GFP) from the Pacific Northwestjellyfish, Aequorea Victoria, has been used extensively in molecular andcell biology as a fluorescent marker. It is a 238 amino acid proteinthat generates its own fluorescent chromophore. The spontaneousgeneration of the chromophore is achieved by cyclization of the internalSer65-Tyr66-Gly67 sequence followed by oxidation of Tyr 66 in thepresence of molecular oxygen (Heim et al., Proc. Natl. Acad. Sci. USA91:12501-12504, 1994). The overall fold of the protein consists of an11-stranded β-barrel capped by α-helices at both ends and contains acoaxial α-helix from which the chromophore is generated (Brejc et al.,Proc. Natl. Acad. Sci. USA 94:2306-2311, 1997; Ormö et al., Science273:1392-1395, 1996; Yang et al., Nat. Biotech. 14:1246-1251, 1996). GFPis unique among light emitting proteins, because it does not require thepresence of any cofactors or substrates for the production of greenlight.

Wild-type GFP has absorption maxima at 398 and 475 nm (Morise et al.,Biochemistry 13:2656-2662, 1974). Excitation at either of thesewavelengths leads to emission of green light at 508 nm (Morise et al.,1974). The usefulness of GFP has been greatly enhanced by theavailability of mutants with a broad range of absorption and emissionmaxima (Heim et al., Proc. Natl. Acad. Sci. USA 91:12501-12504, 1994;Ormö et al., Science 273:1392-1395, 1996). These mutants have madepossible multicolor reporting of cellular processes by allowing for thesimultaneous observation of two or more gene products labeled withdifferent colored GFP variants (Rizzuto et al., Curr. Biol. 6:183-188,1996). In addition, fluorescence resonance energy transfer (FRET)experiments using different colored GFP's have been used to studyprotein-protein interactions in vivo (Heim et al., Curr. Biol.6:178-182, 1996; Mitra et al., Gene 173:13-17, 1996).

More recently, GFP variants have been shown to be sensitive to pH(Wachter et al., Biochemistry 36:9759-9765, 1997; Elsliger et al.,Biochemistry 38:5296-5301, 1999). As a consequence, they have been usedas noninvasive intracellular pH indicators. For instance, Kneen et al.employed the GFP mutant S65T/F64L to determine the pH of the cytoplasmof CHO and LLC-PK1 cell lines (Kneen et al., Biophys. J. 74:1591-1599,1998). Since GFP is genetically encoded, it can be specifically targetedto various subcellular compartments, which is a task not possible withsmall molecule fluorescent dyes (Llopis et al., Proc. Natl. Acad. Sci.USA 95:6803-6808, 1998). Therefore, Llopis and co-workers used the GFPvariant S65G/S72A/T302Y/H231L, which has an increased pK_(a), to measurethe alkaline pH of mitochondria, golgi, and the cytosol of HeLa cellsand rat neonatal cardiomyocytes (Llopis et al., 1998). These reportswere the first to show that GFP variants could be used as biosensors andnot just simple fluorescent markers. However, more recently GFP has beenshown to be sensitive to halide ions and through a fusion withcalmodulin, GFP's fluorescence can also vary in response to calcium ionconcentration (Wachter et al., Curr. Biol. 9:R628-R629, 1999; Miyawakiet al., Proc. Natl. Acad. Sci. USA 96:2135-2140, 1999).

Oxidation-reduction (redox) processes are very important in livingorganisms. The formation of disulfide bonds during protein foldingrelies upon a well maintained redox buffering system of glutathione andoxidized glutathione (Carothers et al., Arch. Biochem. Biophys.268:409425, 1989). There also exists a thioredoxin-like family ofenzymes that catalyze the formation and isomerization of disulfide bondsin proteins (Debarbieux and Beckwith, Cell 99:117-119, 1999). Inaddition, redox signaling during apoptosis has been implicated inactivating mitochondrial permeability transition, leading to cytochromec release (Hall, Eur. J. Clin. Invest. 29:238-245, 1999). Redox changesin the form of cellular oxidation have also been suggested to be a finalstep in the apoptotic process leading to degradation of apoptotic bodies(Cai and Jones, J. Bioenerg. Biomemb. 31:327-334, 1999). Given theimportance of in vivo processes such as protein folding and apoptosisthat are dependant upon redox status, a non-invasive, convenient methodfor studying redox changes within living cells is needed.

Current methods of determining in vivo redox status have manylimitations. Many present techniques require cells to be harvestedbefore their contents can be analyzed. This type of procedure is notonly very invasive but is also not a very accurate measure of the invivo state of the cells. Moreover, it would be impossible with thistechnique to monitor redox changes within the same cell over a period oftime. Recently, Keese et al. (Keese et al., FEBS Lett. 447:135-138,1999) have developed an indicator of redox state in which glutathionereductase crystals were microinjected into the cytosol of humanfibroblasts, and by detecting a color change of the crystals, they wereable to determine the redox potential of the cytosol to be more reducingthan −0.270 V. While this method may allow redox determination withinsingle living cells, the cumbersome nature of the technique is still amajor drawback. The most reasonable protocol for determining redoxstatus is probably still that of Hwang et al. (Hwang et al., Science257:1496-1502, 1992). They employed the tetrapeptideN-Acetyl-Asn-Tyr-Thr-Cys-NH₂ to measure the ratio of thiol to disulfidein the cytosol and secretory pathway of cultured cells. They concludedthat the cytosol is more reducing than the secretory pathway with anapproximate redox potential of −0.221 to −0.236 V for the cytosolcompared to −0.170 to −0.185 V for the secretory pathway. However, thismethod still required harvesting of the cells and like all the othermethods, it is very labor intensive. Moreover, this technique determinedredox potentials based only upon the ratio of reduced glutathione (GSH)to oxidized glutathione (GSSG), potentially ignoring other redoxbuffering components.

SUMMARY OF THE DISCLOSURE

To overcome disadvantages of available methods for determining redoxstatus in cells, GFP mutants (also referred to as redox sensitive GFP(rosGFP) variants) have been designed and are described herein, whichcan detect or “sense” changes in oxidation-reduction potentials. TherosGFP variants have been engineered to have two cysteine amino acidsnear the chromophore and within disulfide bonding distance of eachother.

Examples of the provided GFP variants have ratiometric dual-excitationfluorescent properties as a function of redox state, with apparent redoxpotentials of −0.272 to −0.299 V.

Specific embodiments include rosGFP mutants that differ from wild-typeGFP in that they comprise at least the following amino acidsubstitutions:

-   -   (a) S 147C/Q204C    -   (b) S65T/S147C/Q204C    -   (c) N149C/S202C    -   (d) S65T/N149C/S202C    -   (e) S 147C/N 149C/S202C/Q204C    -   (f) S65T/S147C/N149C/S202C/Q204C

The rosGFP mutants that include the S65T substitution are sensitive topH as well as redox status. Particular provided mutation proteinsinclude those referred to herein as rosGFP1, rosGFP2, rosGFP3, rosGFP4,rosGFP5, and rosGFP6.

Also provided are nucleic acid molecules encoding rosGFPs, including thespecific listed rosGFPs. Optionally, these nucleic acid molecules can befunctionally linked to expression control sequence(s) (such as apromoter), and/or integrated into a vector. Nucleic acid moleculesencoding a rosGFP can be used to transform host cells (such asbacterial, plant, or animal cells); such transformed cells are alsoprovided.

The disclosure also provides methods of using rosGFPs to analyze theredox status of a cell, or a subcellular compartment within a cell. Incertain embodiments, both the redox status and pH of the cell (orsubcellular compartment or other environment) are monitoredconcurrently.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a fluorescence spectra graph, which shows how the fluorescenceof rosGFP2 varies in response to changes in redox potential. The spectrashow two excitation peaks, one near 400 nm and the other at about 490nm, with a clear isosbestic point separating them.

FIG. 2 shows the titration of rosGFP2 with dithiothreitol. The apparentredox potential is −0.279 volts.

FIG. 3 is a graph showing the in vivo redox changes in fluorescenceintensity of rosGFP2, in response to the addition of vitamin K₃. After30 total minutes, the addition of dithiothreitol elicits the oppositeresponse as indicated by the reduced ratio.

FIG. 4 shows an SDS-PAGE analysis that reveals the intracellulardisulfide linkage in rosGFP2. Lanes 1-6, control (C48S/S65T) and lanes8-13 rosGFP2 were incubated with 1 μM CuCl2 (with or without 2 mMN-ethylmaleimide; lanes 2, 3, 11, 12) or with 1 mM DTT (with or without2 mM N-ethylmaleimide; lanes 5, 6, 8, 9). Lane 7 shows approximatemolecular weights in kDa. Lanes 4 and 10 were empty.

FIG. 5. Absorbance and fluorescence excitation spectra of rosGFP2 atvarious redox states. The absorbance spectra (A) show the conversion ofthe neutral (band A; 400 nm) to the anionic (band B; 490 nm) chromophorespecies over time in the presence of 1 mM DTT. Band A is maximized underoxidizing conditions, whereas band B is favored under reducingconditions. Fluorescence spectra (B) were collected at various redoxpotentials and also show the interconversion of chromophore chargestates. Absorbance and fluorescence spectra were both normalized to theintensity of the fully reduced protein.

FIG. 6. Fluorescence excitation spectra of rosGFP4 as a function ofredox potential. The entire spectrum (A) shows the redox potentialdependence on the excitation spectra of rosGFP4. Expanded the regionaround 400 nm (B), reveals a well resolved isosbestic point.Fluorescence intensity values were normalized to the maximum intensityat E_(o)′ −0.320 V and emission was monitored at 510 nm.

FIG. 7. Fluorescence excitation spectra of rosGFP6 as a function ofredox potential. Fluorescence intensity values were normalized to themaximum intensity at E_(o)′ −0.310 V and emission was monitored awayfrom the peak at 535 nm.

FIG. 8. Absorbance and fluorescence excitation spectra of oxidizedrosGFP2 as a function of pH. Absorbance scans (A) were taken on samplesof rosGFP2 containing 0.5 μM CuCl₂ at the indicated pHs. These sampleswere then diluted in the same buffer and their fluorescence excitationspectra (B) were collected. Fluorescence intensity values werenormalized to the maximum intensity at pH 9.0 and emission was monitoredat 510 nm.

FIG. 9. pH titration of oxidized and reduced rosGFP2. Absorbance valuesat 490 nm (band B) were plotted versus pH for oxidized (A) and reduced(B) rosGFP2. The data were then fitted to a titration curve with asingle pK_(a) value.

FIG. 10. Absorbance and fluorescence excitation spectra of reducedrosGFP2 as a function of pH. Absorbance scans (A) were taken on samplesof rosGFP2 containing 1 mM DTT at the indicated pHs. These samples werethen diluted in the same buffer and their fluorescence excitationspectra (B) were collected. Fluorescence intensity values werenormalized to the maximum intensity at pH 9.0 and emission was monitoredat 510 nm. Absorbance readings around 280 nm are greatly altered due tothe presence of DTT and thus are not shown.

FIG. 11. Fluorescence excitation spectra of rosGFP1 at various redoxpotentials. Fluorescence intensity values were normalized to the maximumintensity at E_(o)′ −0.320 V and emission was monitored at 510 nm.

FIG. 12. Fluorescence excitation spectra of rosGFP3 at various redoxpotentials. The entire spectrum (A) shows the redox potential dependenceon the excitation spectra of rosGFP3. Expanded the region around 405 nm(B), reveals the existence of an isosbestic point. Fluorescenceintensity values were normalized to the maximum intensity at E_(o)′−0.330 V and emission was monitored at 510 nm.

FIG. 13. Fluorescence excitation spectra of rosGFP5 at various redoxpotentials. Fluorescence intensity values were normalized to the maximumintensity at E_(o)′ −0.330 V and emission was monitored off the peak at535 nm.

FIG. 14. A fluorescence excitation ratio results in the cancellation ofpH artifacts. In the oxidized (A) or reduced (B) state, a ratio offluorescence intensities at various excitation wavelengths of rosGFP2 isindependent of pH.

FIG. 15. Dual-emission characteristics of rosGFP2. Excitation at 400 nmresults in emission peaks centered near 450 and 510 nm, which have anopposite response to pH changes.

FIG. 16. A fluorescence emission ratio results in the cancellation ofredox potential changes on pH determination. The fluorescence emissionspectra (A) of rosGFP2 were collected at various redox potentials(ratios of DTT and DTT_(ox)) and at a constant pH of 6.0. Plotting theratio of the two emission peaks results in a constant ratio over a largerange of redox states (B). The dashed lines in B represent the maximumand minimum ratios to illustrate the possible dynamic range of rosGFP2as a function of pH.

FIG. 17. A fluorescent micrograph showing the reticular localizationpattern of rosGFPI expressed in the mitochondrial matrix of an in vitrocultured HeLa cell, via fusion at the DNA level to the mitochondrialtargeting sequence of the E₁α subunit of pyruvate dehydrogenase.

FIG. 18. Response of rosGFP1 to H₂O₂ and DTT induced redox potentialchanges in HeLa cell mitochondria. H₂O₂ and DTT were added at theindicted time points to a final concentration of 1 mM and 30 mM,respectively. The Fluorescence Intensity axis corresponds to theindividual wavelengths, whereas the Ratio 400/480 axis corresponds tothe ratio of the two wavelength channels.

FIG. 19. NADH-dependent reduction of rosGFP1 via lipoamidedehydrogenase. Each bar represents the percent reduction of oxidizedrosGFP1 by 1-2 μL LDH, 1 mM lipoate, 1 mM NADH, and/or 1 mM DTT. Sampleswere equilibrated at 22° C. for one hour after which the fluorescenceexcitation was scanned from 325 to 525 nm. Percent reduction values weredetermined by the fluorescence at 490 nm with 100% corresponding toreduction by DTT.

FIG. 20. Fluorescence excitation spectra of rosGFP2 at varyingconcentrations of DTT_(red) and DT_(ox). Fluorescence emission intensitywas monitored at 510 nm and normalized to the maximum intensity of thefully reduced spectrum (solid line).

FIG. 21. Redox equilibrium titration of rosGFP2 with dithiothreitol. Therelative amount of reduced rosGFP2 at equilibrium (R) was measured usinga ratio of the rosGFP2 fluorescence at 510 nm (excitation 490:425 nm).Oxidized rosGFP2 (1 μM) was incubated for four hours in 75 mM HEPES (pH7.0), 140 mM NaCl, and 1 mM EDTA, containing varying ratios of DTT_(red)to DTT_(ox) (1 mM total). The equilibrium constant was determined byfitting the data according to equation 3. After nonlinear regression, aK_(eq) of 2.05×10⁻² was obtained (correlation coefficient: 0.998).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

-   -   SEQ ID NO: 1 shows the amino acid sequence of wild-type GFP.    -   SEQ ID NO: 2 shows the nucleic acid and amino acid sequence of        rosGFP2.    -   SEQ ID NO: 3 shows the amino acid sequence of rosGFP2.    -   SEQ ID NO: 4 shows the nucleic acid and amino acid sequence of        rosGFP1.    -   SEQ ID NO: 5 shows the amino acid sequence of rosGFP1.    -   SEQ ID NO: 6 shows the nucleic acid and amino acid sequence of        rosGFP4.    -   SEQ ID NO: 7 shows the amino acid sequence of rosGFP4.    -   SEQ ID NO: 8 shows the nucleic acid and amino acid sequence of        rosGFP3.    -   SEQ ID NO: 9 shows the amino acid sequence of rosGFP3.    -   SEQ ID NO: 10 shows the nucleic acid and amino acid sequence of        rosGFP6.    -   SEQ ID NO: 11 shows the amino acid sequence of rosGFP6.    -   SEQ ID NO: 12 shows the nucleic acid and amino acid sequence of        rosGFP5.    -   SEQ ID NO: 13 shows the amino acid sequence of rosGFP5.    -   SEQ ID NO: 14 shows the amino acid sequence of a tetrapeptide        used to measure the ratio of thiol to disulfide in the cytosol        and secretory pathway of cultured cells.    -   SEQ ID NO: 15 shows the amino acid sequence of a nuclear        localization sequence.    -   SEQ ID NO: 16 shows the amino acid sequence of a mitochondrion        localization sequence.    -   SEQ ID NO: 17 shows the amino acid sequence of an endoplasmic        reticulum localization sequence.

DETAILED DESCRIPTION

I. Abbreviations

GFP green fluorescent protein

rosGFP redox-sensitive GFP

wtGFP wild-type GFP

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-2182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments, the followingexplanations of specific terms are provided:

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide substantially encoded by an immunoglobulin geneor immunoglobulin genes, or fragments thereof, which specifically bindsand recognizes an analyte (antigen). Immunoglobulin genes include thekappa, lambda, alpha, gamma, delta, epsilon and mu constant regiongenes, as well as the myriad immunoglobulin variable region genes.

Antibodies exist, e.g., as intact immunoglobulins or as a number ofwell-characterized fragments produced by digestion with variouspeptidases. For instance, FAbs, Fvs, and single-chain Fvs (SCFvs) thatbind to GFP would be GFP-specific binding agents. Antibody fragments aredefined as follows: (1) Fab, the fragment which contains a monovalentantigen-binding fragment of an antibody molecule produced by digestionof whole antibody with the enzyme papain to yield an intact light chainand a portion of one heavy chain; (2) Fab′, the fragment of an antibodymolecule obtained by treating whole antibody with pepsin, followed byreduction, to yield an intact light chain and a portion of the heavychain; two Fab′ fragments are obtained per antibody molecule; (3)(Fab′)2, the fragment of the antibody obtained by treating wholeantibody with the enzyme pepsin without subsequent reduction; (4)F(ab′)2, a dimer of two Fab′ fragments held together by two disulfidebonds; (5) Fv, a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; and (6) single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain, the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chainmolecule. The term “antibody,” as used herein, also includes antibodyfragments either produced by the modification of whole antibodies orthose synthesized de novo using recombinant DNA methodologies.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA mayalso contain untranslated regions (UTRS) that are responsible fortranslational control in the corresponding RNA molecule. cDNA is usuallysynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Conservative variations: Variants of a particular nucleic acid sequence,which encode identical or essentially identical amino acid sequences.Because of the degeneracy of the genetic code, a large number offunctionally identical nucleic acids encode any given polypeptide. Forinstance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode theamino acid arginine. Thus, at every position where an arginine isspecified within a protein encoding sequence, the codon can be alteredto any of the corresponding codons described without altering theencoded protein. Such nucleic acid variations are “silent variations,”which are one species of conservative variations. Each nucleic acidsequence herein that encodes a polypeptide also describes every possiblesilent variation. The genetic code is shown in Table 1. TABLE 1 Firstposition Second position Third position (5′ end) U C A G (3′ end) U PheSer Tyr Cys U Phe Ser Tyr Cys C Leu Ser Stop Stop A Leu Ser Stop Trp G CLeu Pro His Arg U Leu Pro His Arg C Leu Pro Gln Arg A Leu Pro Gln Arg GA Ile Thr Asn Ser U Ile Thr Asn Ser C Ile Thr Lys Arg A Met Thr Lys ArgG G Val Ala Asp Gly U Val Ala Asp Gly C Val Ala Glu Gly A Val Ala GluGly G

One of skill will recognize that each codon in a nucleic acid (exceptAUG, which is rdinarily the only codon for methionine) can be modifiedto yield a functionally identical olecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid that ncodes apolypeptide is implicit in each described sequence.

Furthermore, one of ordinary skill will recognize that individualsubstitutions, deletions or additions which alter, add or delete asingle amino acid or a small percentage of amino acids (for instanceless than 5%, in some embodiments less than 1%) in an encoded sequenceare conservative variations where the alterations result in thesubstitution of an amino acid with a chemically similar amino acid.

Conservative amino acid substitutions providing functionally similaramino acids are well known in the art. The following six groups eachcontain amino acids that are conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).        Not all residue positions within a protein will tolerate an        otherwise “conservative” substitution. For instance, if an amino        acid residue is essential for a function of the protein, even an        otherwise conservative substitution may disrupt that activity.        By way of example, in a GFP the residues that compose the        chromophore do not generally tolerate amino acid substitutions.

Epitope tags: Short stretches of amino acids to which a specificantibody can be raised, which in some embodiments allows one tospecifically identify and track the tagged protein that has been addedto a living organism or to cultured cells. Detection of the taggedmolecule can be achieved using a number of different techniques.Examples of such techniques include: immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (“western”), and affinity chromatography. Examplesof useful epitope tags include FLAG, T7, HA (hemagglutinin) and myc.

Expression control sequence: This phrase refers to nucleotide sequencesthat regulate the expression of a nucleotide sequence to which they areoperatively linked. Expression control sequences are “operativelylinked” to a nucleotide sequence when the expression control sequencescontrol and regulate the transcription and, as appropriate, translationof the nucleotide sequence. Thus, expression control sequence(s) caninclude promoters, enhancers, transcription terminators, a start codon(i.e., ATG) in front of a protein-encoding sequence, intron splicingsignals, and stop codons.

Fluorescent property: A characteristic of a fluorescent molecule.Examples of fluorescent properties include the molar extinctioncoefficient at an appropriate excitation wavelength, the fluorescencequantum efficiency, the shape of the excitation spectrum or emissionspectrum (the “fluorescence spectrum”, the excitation wavelength maximumand emission wavelength maximum, the ratio of excitation amplitudes attwo different wavelengths, the ratio of emission amplitudes at twodifferent wavelengths, the excited state lifetime, or the fluorescenceanisotropy. A measurable difference in any one of these propertiesbetween wild-type Aequorea GFP and the mutant form is useful. Ameasurable difference can be determined by determining the amount of anyquantitative fluorescent property, e.g., the amount of fluorescence at aparticular wavelength, or the integral of fluorescence over the emissionspectrum. Determining ratios of excitation amplitude or emissionamplitude at two different wavelengths (“excitation amplitude ratioing”and “emission amplitude ratioing,” respectively) for a particularmolecule are advantageous. The ratioing process provides an internalreference and cancels out variations, for instance in the absolutebrightness of the excitation source, the sensitivity of the detector,and light scattering or quenching by the sample.

Fusion protein: Proteins that have two (or more) parts fused together,which are not found joined together in nature. In general, the twodomains are genetically fused together, in that nucleic acid moleculesthat encode each protein domain are functionally linked together, forinstance by a linker oligonucleotide, thereby producing a singlefusion-encoding nucleic acid molecule. The translated product of such afusion-encoding nucleic acid molecule is the fusion protein.

Green fluorescent protein (GFP): GFP is a 238 amino acid, spontaneouslyfluorescent protein, originally isolated from the Pacific Northwestjellyfish Aequorea victoria. The amino acid sequence of wtGFP is shownin SEQ ID NO: 1. This protein has become an extremely popular tool inmolecular and cell biology (for reviews: Tsien, Annu. Rev. Biochem.67:509-544, 1998; Remington, In Bioluminescence and chemiluminescence(eds. T. O. Baldwin and M. M. Sigler), pp. 195-211, 2000, Academic, SanDiego, Calif.). Originally GFP was used as a passive indicator of geneexpression and protein localization. More recently, GFP has taken on therole of an active indicator of such things as intracellular H⁺, Ca²⁺,and halide ion concentrations (Kneen et al., Biophys. J. 74:1591-1599,1998; Llopis et al., Proc. Natl. Acad. Sci. USA 95:6803-6808, 1998;Baird et al., Proc. Natl. Acad. Sci. USA 96:11241-11246, 1999; Jayaramanet al., J. Biol. Chem. 275:6047-6050, 2000).

In addition to GFP being highly fluorescent, protease resistant, andvery stable throughout a wide range of pH and solvent conditions, italso has the advantage of being functional as a single protein encodedby a single gene. These traits result in a biological probe moleculethat can be expressed in nearly all organisms. It also can be targetedto subcellular organelles by a host cell, for instance through theinclusion of a targeting sequence on the construction from which it isexpressed. GFP is a non-invasive indicator, which allows for experimentsto be conducted and monitored in a single cell over a period of time.

GFPs as discussed herein (including rosGFPs) can be expressed as fusionproteins. The GFP protein can be functionally fused to, for instance, atag (such as an epitope tag), a targeting molecule (such as a targetingpeptide), or a protein (or fragment thereof) that provides an additionalfunction, such as a biochemical, biological, or localization function.The construction and production of fusion proteins is well known to oneof ordinary skill in the art.

A “mutant” GFP is a green fluorescent protein (or nucleic acid encodingsuch) that has at least one residue that is different from (mutatedfrom) the wtGFP. Mutations include, for instance, conservative ornon-conservative amino acid substitutions, silent mutations (wherein thenucleic acid sequence is different from wild-type at a particularresidue, but the amino acid sequence is not), insertions (includingfusion proteins), and deletions. Myriad mutant GFPs are known, includingfor instance those disclosed in the following patent documents: U.S.Pat. Nos. 5,804,387; 6,090,919; 6,096,865; 6,054,321; 5,625,048;5,874,304; 5,777,079; 5,968,750; 6,020,192; and 6,146,826; and publishedinternational patent application WO 99/64592.

Specific examples of mutant GFPs include proteins in which thefluorescence spectrum of the mutant is responsive to an environmentalvariable, such as temperature, proton concentration (pH), saltconcentration, and redox status. Particular mutant GFPs as providedherein are sensitive to redox status, and others are responsive to bothredox status and pH. A fluorescence spectrum is “responsive” to anenvironmental variable if the spectrum changes with changes in thatvariable.

Immunoassay: An assay that utilizes an antibody to specifically bind ananalyte. The immunoassay is characterized by the use of specific bindingproperties of a particular antibody to isolate, target, detect, and/orquantify the analyte, or alternately using a particularly analyte (e.g.,an antigen) to isolate, target, detect, and/or quantify the antibody.

In vitro amplification: Techniques that increases the number of copiesof a nucleic acid molecule in a sample or specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to nucleic acid template in the sample. Theprimers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of in vitroamplification may be characterized by electrophoresis, restrictionendonuclease cleavage patterns, oligonucleotide hybridization orligation, and/or nucleic acid sequencing, using standard techniques.Other examples of in vitro amplification techniques include stranddisplacement amplification (see U.S. Pat. No. 5,744,311);transcription-free isothermal amplification (see U.S. Pat. No.6,033,881); repair chain reaction amplification (see WO 90/01069);ligase chain reaction amplification (see EP-A-320 308); gap fillingligase chain reaction amplification (see U.S. Pat. No. 5,427,930);coupled ligase detection and PCR (see U.S. Pat. No. 6,027,889); andNASBA™ RNA transcription-free amplification (see U.S. Pat. No.6,025,134).

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Label: A composition detectable by spectroscopic, photochemical,biochemical, immunochemical, or chemical means. For example, usefullabels include ³²p (or other radio-isotope), fluorescent dyes,fluorescent proteins, electron-dense reagents, enzymes (e.g., for use inan ELISA), biotin, dioxigenin, or haptens and proteins or peptides forwhich antisera or monoclonal antibodies are available. A label oftengenerates a measurable signal, such as radioactivity, fluorescent lightor enzyme activity, which can be used to detect and/or quantitate theamount of labeled molecule.

Nucleic acid: A deoxyribonucleotide or ribonucleotide polymer in eithersingle-or double-stranded form. Unless otherwise limited, this termencompasses known analogs of natural nucleotides that can function in asimilar manner as naturally occurring nucleotides. When a nucleic acidmolecule is represented herein by a DNA sequence, the corresponding RNAmolecules are likewise understood, in which “U” replaces “T.”

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame. Similarly, two peptide or polypeptide sequences areconsidered to be operably linked if they are linked to each other insuch a way that they function in the intended manner.

Polypeptide or Protein: A polymer of amino acid residues. The termsapply to amino acid polymers in which one or more amino acid residue isan artificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterm “recombinant protein” refers to a protein that is produced byexpression of a nucleotide sequence (which encodes the protein) from arecombinant DNA molecule.

Preferred mammalian codon(s): The subset of codons from among the set ofall possible codons encoding an amino acid that are most frequently usedin proteins expressed in mammalian cells. Table 2 summarizes thepreferred mammalian codons for each amino acid: TABLE 2 Amino AcidPreferred codons* Gly GGC, GGG Glu GAG Asp GAC Val GUG, GUC Ala GCC, GCUSer AGC, UCC Lys AAG Asn AAC Met AUG Ile AUC Thr ACC Trp UGG Cys UGC TyrUAU, UAC Leu CUG Phe UUC Arg CGC, AGG, AGA Gln CAG His GAC Pro CCC

Primers: Primers are short nucleic acid molecules, for instance DNAoligonucleotides 10 nucleotides or more in length. Longer DNAoligonucleotides may be about 15, 20, 25, 30 or 50 nucleotides or morein length. Primers can be annealed to a complementary target DNA strandby nucleic acid hybridization to form a hybrid between the primer andthe target DNA strand, and then the primer extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

Methods for preparing and using nucleic acid primers are described, forexample, in Sambrook et al. (In Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989), Ausubel et al. (ed.) (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998), and Innis et al.(PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990). Amplification primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, ® 1991, WhiteheadInstitute for Biomedical Research, Cambridge, Mass.). One of ordinaryskill in the art will appreciate that the specificity of a particularprobe or primer increases with its length.

Probes: A probe comprises an isolated nucleic acid attached to adetectable label or other reporter molecule. Typical labels includeradioactive isotopes, enzyme substrates, co-factors, ligands,chemiluminescent or fluorescent agents, haptens, and enzymes. Methodsfor labeling and guidance in the choice of labels appropriate forvarious purposes are discussed, e.g., Sambrook et al. (In MolecularCloning: A Laboratory Manual, CSHL, New York, 1989) and Ausubel et al.(In Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

Promoter: A promoter is an ordered set of nucleic acid control sequencesthat direct transcription of a nucleic acid. A promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elementsthat can be located as much as several thousand base pairs from thestart site of transcription.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell or within aproduction reaction chamber (as appropriate).

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Recombinant host cell: A cell (such as a bacterial, plant, or animalcell) that comprises a recombinant nucleic acid molecule. Thus, forexample, recombinant host cells can express genes and/or proteins thatare not found within the native (non-recombinant) form of the cell.

Redox status: A measurement of the oxidation-reduction (redox) potentialof an environment, for instance the environment within a cell or asubcellular compartment.

Fundamentally, redox reactions are a family of reactions that areconcerned with the transfer of electrons between species. Oxidationrepresents a loss of electrons, reduction a gain of electrons.Oxidation-reduction reactions always occur together, and the electronsgained by the molecule that is reduced must balance those given up bythe substance that is oxidized. The oxidation-reduction potential (orredox status) of a solution is a measurement of the oxidation orreduction force of the solution, and is indicative of the oxidation orreduction ability.

Redox status of any solution can be measured. For instance, the redoxstatus of the solution within a cell (i.e., the cytosol) can be analyzedusing the provided rosGFPs. Similarly, the redox status of the solutionwithin a subcellular organelle (such as the nucleus, mitochondria,plastid, vacuole, secretory pathway compartment and so forth) can beanalyzed.

Stringent conditions: A set of temperature and ionic conditions used ina nucleic acid hybridization. Stringent conditions are sequencedependent and are different under different environmental parameters.Generally, stringent conditions are selected to be about 5° C. to 20°C., lower than the thermal melting point (T_(m)) for the specific targetsequence. The T_(m) is the temperature (under defined ionic strength andpH) at which 50% of the target sequence hybridizes to a perfectlymatched (100% identical) probe molecule.

Substantially identical/similar: An amino acid sequence or a nucleotidesequence is substantially identical (or substantially similar) to areference sequence if the amino acid sequence or nucleotide sequence hasat least 80% sequence identity with the reference sequence over a givenwindow of comparison. Thus, substantially similar sequences includethose having, for example, at least 85% sequence identity, at least 90%sequence identity, at least 95% sequence identity or at least 99%sequence identity. Two sequences that are 100% identical to each otherare, of course, also substantially identical.

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of naked DNA by electroporation, lipofection, andparticle gun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication. A vector may also include one or more selectable markergenes and other genetic elements known in the art.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Comprises means includes. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

III. Redox-Sensitive Green Fluorescent Proteins

Many fundamental biological processes rely upon a properly maintainedintracellular redox environment (Cuozzo and Kaiser, Nat. Cell Biol.1:130-135, 1999; Debarbieux and Beckwith, Cell 99:117-119, 1999; Hall,Eur. J. Clin. Invest. 29:238-245, 1999; Cai and Jones, J. Bioenerg.Biomemb. 31:327-334, 1999). Moreover, reactive oxygen species likeO₂.⁻HO., or H₂O₂, arise in cells by a variety of processes includinglight, radiation, or the respiratory chain. These radical speciespresent major threats to organisms by damaging DNA, membranes, or othercellular components.

The importance of redox status to biological process might imply thatresearchers have numerous well-established techniques for monitoringredox potentials in vivo. However, this is not the case. Currentredox-sensing methods are invasive, require large sample sizes, arelabor intensive, and do not allow for real-time determinations on livingcells.

This disclosure describes a new class of GFP variants (rosGFPs) thatdisplay ratiometric excitation properties as a function of redoxpotential. rosGFP biosensors allow for real-time measurements of redoxstatus on small groups of cells, individual cells, or even withincertain cellular organelles. As a result of being genetically encoded,these molecules are non-invasive, and their fluorescence characteristicsthey are easy to use as sensing molecules.

To create a redox sensor that overcomes many of the drawbacks of othermethods of in vivo redox status determination, site-directed mutagenesiswas carried out on the green fluorescent protein (GFP). By mutatingresidues near the chromophore in GFP to cysteines, novel redox-sensitiveGFPs (rosGFPs) were constructed. They display ratiometricdual-excitation fluorescent properties as a function of redox state,with apparent redox potentials of −0.272 to −0.299 V.

Unexpectedly, these rosGFPs also exhibited ratiometric dual-emissionproperties in response to pH changes, providing the unique possibilityto simultaneously monitor redox potential and pH changes with the sameprobe. T65S reversion leads to pH-independent redox sensors with alteredredox potentials and large UV excitation peaks, which can be used toovercome background levels of cellular auto-fluorescence.

Crystal structure analyses of an oxidized and reduced rosGFP to 1.9 and2.0 Å, respectively, indicate that changes in the structure on reductionor oxidation of the disulfide bridge could account for the observedspectral changes.

As shown herein using cultured HeLa cells, rosGFPs can reversiblyrespond to exogenous redox stimuli and have redox potentials near thatof mitochondria, with the apparent redox potential of mitochondriaestimated to more reducing than about −0.30 V. The apparent redoxpotential of the cytosol also was found to be more reducing than −0.30V.

One example redox sensitive GFP mutant (rosGFP2) differs from wild-typeGFP in that it contains the following amino acid residue mutations:C48S/S65T/Q80R/S147C/Q204C (SEQ ID NOs: 2 and 3). FIG. 1 illustrates howthe fluorescence of rosGFP2 varies in response to changes in redoxpotential. The spectra show two excitation peaks, one near 400 nm andthe other at about 490 nm, with a clear isosbestic point separatingthem. These dual excitation peaks respond in opposite directions toredox potential, making this indicator ratiometric for redox potential.Ratiometric indicators are known to reduce or eliminate distortions ofdata caused by photobleaching, indicator concentration, variable cellthickness, illumination stability, excitation pathlength, andnon-uniform indicator distribution within cells or between groups ofcells (Grynkiewicz et al., J. Biol. Chem. 260:3440-3450, 1985).

By monitoring the chromophore fluorescence at the 490 nm excitation peakas a function of redox potential, the apparent redox potential ofrosGFP2 was found. FIG. 2 shows the titration of rosGFP2 withdithiothreitol. From this plot the apparent redox potential is −0.279volts. To demonstrate that the redox-sensitive probe responds to in vivoredox changes, it was targeted to the mitochondria of HeLa cells. Asseen in FIG. 3, the starting ratio of the 400 to 490 nm peak inmitochondria is around 0.28. After the addition of the redox-cyclingagent vitamin K₃, the ratio dramatically increases to about 0.42. Thisincrease in the ratio is indicative of an oxidation event that is to beexpected according to the reaction catalyzed by vitamin K₃ (Suttie,Annu. Rev. Biochem. 54:459-477, 1985). After 30 total minutes, theaddition of dithiothreitol elicits the opposite response as indicated bythe smaller ratio.

Characteristics of several redox-sensitive GFP mutants are summarized inTable 3. TABLE 3 Mutant Excitation Redox name Substitutions PeaksPotential* rosGFP2 C48S/565T/S147C/Q204C 400 nm −0.272 (SEQ ID NOs: 2and 3) 490 nm rosGFP1 C48S/S147C/Q204C 397 nm −0.288 (SEQ ID NOs: 4 and5) 477 nm rosGFP4 C48S/S65T/N149C/S202C 400 nm −0.286 (SEQ ID NOs: 6 and7) 490 nm rosGFP3 C48S/N149C/S202C 392 nm −0.299 (SEQ ID NOs: 8and 9)475 nm rosGFP6 C48S/S65T/S147C/N149C/S202C/ 398 nm −0.280 Q204C (SEQ IDNOs: 10 and 11) 490 nm rosGFP5 C48S/S147C/N149C/S202C/Q204C 395 nm−0.296 (SEQ ID NOs: 12 and 13) 475 nm*voltsProperties of rosGFPs

Examples of redox-sensitive GFPs described herein have many desirablequalities. In addition to being genetically encoded fluorescentindicators, they are ratiometric. This ratiometric behavior has theadvantage of reducing or eliminating distortions of data caused byphotobleaching, indicator concentration, variable cell thickness,illumination stability, excitation path length, and non-uniformindicator distribution within cells or between groups of cells(Grynkiewicz et al., J. Biol. Chem. 260:3440-3448, 1985; Whitaker etal., Anal. Biochem. 194:330-344, 1991). In addition, a ratio ofexcitation wavelengths greatly minimizes the contribution of pHartifacts to the fluorescent signal over a pH range from 6 to 8.5.Similarly, a ratio of emission wavelengths can result in pH monitoringwithout artifacts introduced by the redox state of the biosensor.

The second-generation rosGFPs, with more closely matched excitation peakamplitudes, may aid in fluorescence microscopy experiments by allowingthe same camera/detector settings to capture both images that constitutethe ratio. Furthermore, second-generation rosGFPs have a larger 400 nmfluorescence amplitude, which is desirable for detection of the rosGFPprobe over background levels of cellular autofluorescence.

Accuracy of Standard Redox Potentials

The main problem in the determination of redox potentials is that E_(o)values cannot be measured directly, but rather are calculated from theknown E_(o) of another redox couple, equilibrated with the redox coupleof unknown E_(o). The standard redox potential can thus vary over alarge range depending on experimental conditions and the choice of redoxcouple.

In this disclosure, the value of −0.323 V for the dithiothreitol couplewas chosen, because it has been very accurately determined in 0.05-0.02M phosphate buffer at pH 7.0 and 30.0°+/−0.5° C. using thelipoamide-lipoamide dehydrogenase couple (Szajewski and Whitesides, J.Am. Chem. Soc. 102:2011-2026, 1980). Because redox potentials are pH andtemperature dependant, all redox equilibria were measured at the same pH(7.0) and temperature (30° C.) as was used to evaluate the standardredox potential of the dithiothreitol couple. Another reason to use thevalue of −0.323 V for the dithiothreitol couple is that the standardpotentials of other commonly used thiol reagents (including the GSH-GSSGcouple) have been determined under identical conditions (Szajewski andWhitesides, J. Am. Chem. Soc. 102:2011-2026, 1980). This will allow forthe direct comparison of redox potentials determined by use of rosGFPsto those determined by other methods, provided the experimentalconditions are reproduced.

For the DTT_(red)-DTT_(ox) couple, the standard potential throughoutliterature is well accepted to be about −0.330 V at near biochemist'sstandard state of pH 7 and 25° C. However, such agreement is not alwaysthe norm. In the case of the glutathione redox couple, very differentvalues for the standard redox potential have been reported. Thepublished values deviate from a somewhat oxidizing value of −0.205 V toa more reducing value of −0.250 V (Szajewski and Whitesides, J. Am.Chem. Soc. 102:2011-2026, 1980; Rost and Rapoport, Nature 201:185, 1964;Torchinsky, Sulfur in proteins. Pergamon Press Ltd., 1981, New York,N.Y.). The 45 mV difference in literature values might be problematicwhen trying to compare redox potentials of rosGFPs to previouslydetermined estimates of the redox potential inside cells.

Even with the large discrepancy in the standard potential of GSH-GSSG,results reported herein indicate that in vivo redox potentials may bemore reducing than previous estimates. Hwang et al., using thetetrapeptide N-Acetyl-Asn-Tyr-Thr-Cys-NH₂ (SEQ ID NO: 14) to measure theratio of thiol to disulfide in the cytosol and secretory pathway ofcultured cells, concluded that the cytosol is more reducing than thesecretory pathway with an approximate redox potential of −0.221 to−0.236 V versus −0.170 to −0.185 V, respectively (Hwang et al., Science257:1496-1502, 1992). In addition, based on prior determinations of theconcentration and ratio of GSH to GSSG in mitochondria, the redoxpotential of this compartment is calculated to be −0.210 to −0.230 V.However, Keese et al. recently developed an indicator of redox state inwhich they transfer glutathione reductase crystals into the cytosol ofliving cells and then detect a color change in the crystals (Keese etal., FEBS Lett. 447:135-138, 1999). Using that method, Keese et al.determined the redox potential of the cytosol of human fibroblasts to bemore reducing than −0.270 V at pH 7.4 and 37° C. That result is inagreement with the estimate reported herein of the cytosolic redoxpotential for HeLa cells being more negative than −0.330 V.

IV. Construction of rosGFPs

The examples below provide specific methods for producing certainembodiment rosGFPs. More generally, in light of the disclosure hereinthat disulfide bonding pairs can be incorporated into GFP to produceredox-sensitive GFP mutants, it will be understood to one of ordinaryskill in the art that many different methods could be used to makerosGFPs.

A. GFP Nucleic Acids and Site-Directed Mutations

DNA encoding wtGFP is available commercially, for example from CLONTECH(Palo Alto, Calif.). Methods of producing mutants containing apredetermined nucleotide sequence are well known to those of ordinaryskill in the art. Two widely used methods are Kunkel mutagenesis and PCRmutagenesis. A detailed description of the two techniques can be foundin Current Protocols in Molecular Biology, Wiley Interscience, 1987,Sections 8.1 and 8.5 respectively. See also Kunkel, Proc. Acad. Nat.Sci. USA 82:488492, 1985; and Saiki et al., Science 239:487-491, 1988.

It is also possible to synthesize mutant GFPs and DNA encoding formutant GFPs directly, by synthetic methods well known in the art. Themutant sequences can be expressed in a variety of systems, includingbacterial, yeast, plant and mammalian cells. DNA encoding the mutant GFPis inserted in an expression vector and transformed into cells ofinterest. The sequence encoding for GFP is inserted in the vector in thecorrect reading frame near a strong promoter, and the expression of GFPis induced. Vectors suitable for specific expression systems are wellknown in the art, and are widely available commercially. Vectors havingcodons optimized for expression in a variety of systems, including yeastand mammalian cells, are also available. For a description of silentnucleotide sequence mutations optimized for mammalian expression, seefor example Haas et al. (Curr. Biol. 6:315-324, 1996).

B. Expression of rosGFPs

One skilled in the art will understand that there are myriad ways toexpress a recombinant protein such that it can subsequently be purified.In general, an expression vector carrying the nucleic acid sequence thatencodes the desired protein will be transformed into a microorganism forexpression. Such microorganisms can be prokaryotic (bacteria) oreukaryotic (e.g., yeast). One appropriate species of bacteria isEscherichia coli (E. coli), which has been used extensively as alaboratory experimental expression system. A eukaryotic expressionsystem will be preferred where the protein of interest (or a domainwithin a fusion protein of interest) requires eukaryote-specificpost-translational modifications such as glycosylation. Also, proteincan be expressed using a viral (e.g., vaccinia) based expression system.Protein can also be expressed in animal cell tissue culture, and such asystem will be appropriate where animal-specific protein modificationsare desirable or required in the recombinant protein.

The expression vector can include a sequence encoding a targetingpeptide, positioned in such a way as to be fused to the coding sequenceof the rosGFP. Targeting domains may allow the fusion protein to betargeted to specific extra-cellular locations, or simply to be secretedfrom the cell, and may be removed during or soon after synthesis of thefusion protein. In addition, multiple targeting peptides can be includedin a single fusion, for instance a peptide/domain that directs thefusion protein to be secreted, and another peptide/domain that directsthe secreted protein to a target (cell, tissue, organ, etc.). Variousappropriate prokaryotic and eukaryotic targeting peptides, and nucleicacid molecules encoding such, are known to one of ordinary skill in theart. Through the use of a eukaryotic secretion-type signal sequence, arosGFP fusion protein can be expressed in a transgenic animal (forinstance a cow, pig, or sheep) in such a manner that the fusion proteinis secreted into the milk of the animal. Targeting protein portions alsomay be used to ensure that a transgenically expressed fusion protein issecreted into the circulatory system of the transgenic animal, therebypermitting the fusion protein to be transported to a target (cell,tissue, organ, etc.).

Optional localization peptide sequences may be, for instance, a nuclearlocalization sequence, an endoplasmic reticulum localization sequence, aperoxisome localization sequence, a mitochondrial localization sequence,or a localized protein. Localization sequences can be targetingsequences which are described, for example, in “Protein Targeting,”chapter 35 of Stryer, Biochemistry (4th ed.). W. H. Freeman, 1995. Thelocalization sequence can also be a localized protein (or domainthereof). Some localization sequences include the following non-limitingexamples (Table 4): TABLE 4 SEQ Subcellular location Localizationsequence(s) ID NO: nucleus KKKRK 15 mitochondrionMLRTSSLFTRRVQPSLFRNILRLQST-* 16 endoplasmic KDEL** (providing retentionin the ER) 17 reticulum when used with a signal sequence* peroxisomeSKF** prenylation or CaaX, CC, CXC, or CCXX** insertion into plasmamembrane cytoplasmic side of fusion to SNAP-25 plasma membrane Golgifusion to furin*amino terminal**carboxy terminal --

Vectors suitable for stable transformation of culturable cells are alsowell known. Typically, such vectors include a multiple-cloning sitesuitable for inserting a cloned nucleic acid molecule, such that it willbe under the transcriptional control of 5′ and 3′ regulatory sequences.In addition, transformation vectors include one or more selectablemarkers; for bacterial transformation this is often an antibioticresistance gene. Such transformation vectors typically also contain apromoter regulatory region (e.g., a regulatory region controllinginducible or constitutive expression), a transcription initiation startsite, a ribosome binding site, an RNA processing signal, and atranscription termination site, each functionally arranged in relationto the multiple-cloning site. For production of large amounts ofrecombinant proteins, an inducible promoter is preferred. This permitsselective production of the recombinant protein, and allows both higherlevels of production than constitutive promoters, and enables theproduction of recombinant proteins that may be toxic to the expressingcell if expressed constitutively.

In addition to these general guidelines, protein expression/purificationkits are produced commercially. See, for instance, the QIAEXPRESS™expression system from QIAGEN (Chatsworth, Calif.) and variousexpression systems provided by INVITROGEN (Carlsbad, Calif.).

C. Purification of rosGFPs

One skilled in the art will understand that there are myriad ways topurify recombinant polypeptides, and such typical methods of proteinpurification may be used to purify the disclosed rosGFPs. Such methodsinclude, for instance, protein chromatographic methods including ionexchange, gel filtration, HPLC, monoclonal antibody affinitychromatography and isolation of insoluble protein inclusion bodies afterover production. In addition, purification affinity-tags, for instance asix-histidine sequence, may be recombinantly fused to the protein andused to facilitate polypeptide purification (e.g., in addition toanother functionalizing portion of the fusion, such as a targetingdomain or another tag). A specific proteolytic site, for instance athrombin-specific digestion site, can be engineered into the proteinbetween the tag and the remainder of the fusion to facilitate removal ofthe tag after purification, if such removal is desired.

Commercially produced protein expression/purification kits providetailored protocols for the purification of proteins made using eachsystem. See, for instance, the QIAEXPRESS™ expression system from QIAGEN(Chatsworth, Calif.) and various expression systems provided byINVITROGEN (Carlsbad, Calif.). Where a commercial kit is employed toproduce a rosGFP, the manufacturer's purification protocol is apreferred protocol for purification of that protein. For instance,proteins expressed with an amino-terminal hexa-histidine tag can bepurified by binding to nickel-nitrilotriacetic acid (Ni-NTA) metalaffinity chromatography matrix (The QIAexpressionist, QIAGEN, 1997).

D. Optional Modifications to rosGFPs

Optionally, redox-sensitive GFP variants/mutants can be “humanized” asdescribed in U.S. Pat. No. 5,874,304, or can contain other mutations(such as substitutions) as dictated by the end-use of the protein.

Also, the mutant GFP proteins can be expressed as part of a fusionprotein. The construction of fusion proteins from domains of knownproteins, or from whole proteins or proteins and peptides, is wellknown. In general, a nucleic acid molecule that encodes the desiredprotein and/or peptide portions are joined using genetic engineeringtechniques to create a single, operably linked fusion oligonucleotide.Appropriate molecular biological techniques may be found in Sambrook etal. (1989). Examples of genetically engineered multi-domain proteins,including those joined by various linkers, and those containing peptidetags, can be found in the following patent documents:

U.S. Pat. No. 5,994,104 (“Interleukin-12 fusion protein”);

U.S. Pat. No. 5,981,177 (“Protein fusion method and construction”);

U.S. Pat. No. 5,914,254 (“Expression of fusion polypeptides transportedout of the cytoplasm without leader sequences”);

U.S. Pat. No. 5,856,456 (“Linker for linked fusion polypeptides”);

U.S. Pat. No. 5,767,260 (“Antigen-binding fusion proteins”);

U.S. Pat. No. 5,696,237 (“Recombinant antibody-toxin fusion protein”);

U.S. Pat. No. 5,587,455 (“Cytotoxic agent against specific virusinfection”);

U.S. Pat. No. 4,851,341 (“Immunoaffinity purification system”);

U.S. Pat. No. 4,703,004 (“Synthesis of protein with an identificationpeptide”); and

WO 98/36087 (“Immunological tolerance to HIV epitopes”).

In particular, patent disclosures related to fusion proteins containinga GFP moiety include the following:

U.S. Pat. No. 6,180,343 (“Green fluorescent protein fusions with randompeptides”);

WO 99/54348 (“Rapidly degrading GFP-fusion proteins and methods ofuse”);

WO 99/19470 (“GFP-annexin fusion proteins”)

WO 98/14605 (“Renilla luciferase and green fluorescent protein fusiongenes”); and

EP 949269 (“Biosensor protein”).

V. Applications

With the provision of redox-sensitive GFP indicators, methods ofmonitoring redox changes in real time in a targeted environment, forinstance in the mitochondria (or other subcellular compartment) ofsingle living cells, are enabled.

Redox-sensitive GFPs mutant are useful in a wide variety ofapplications, including the monitoring of redox status of individualcells or subcellular compartments. Certain of the provided rosGFPs arealso useful for monitoring pH concurrently with redox status, asexplained herein.

Mutant GFPs as described herein are suitable for use as markers fortransformation of mammalian cells. Often, a gene of therapeutic interestdoes not produce an easily distinguishable phenotype in cells expressingthat gene. Thus, such a therapeutic gene can be inserted into a vectorthat contains a marker gene. The therapeutic gene and the marker geneare placed in the vector under the control of a cellular or viralpromoter, and introduced into mammalian cells of interest; subsequently,the transfected cells (the cells containing the vector) are selectedaccording to the phenotype determined by the marker gene. The use of GFPfor selection obviates the need to grow the mammalian cells of interestin the presence of drugs in order to select for the transfected cells.In addition, due to the redox-sensitive nature of the provided GFPmutants, the redox status of the transformed cells (or respectivesubcellular compartment(s)) can be directly measured. Cells transformedwith a nucleic acid comprising a rosGFP can be sorted by FACS.

For the study of protein localization, fusion of a rosGFP mutant and asequence encoding a cellular protein (or encoding a fragment,sub-domain, or domain of a protein), and subsequent expression of thefusion construct, results in a fluorescent fusion protein that islocalized at the normal intracellular location of the protein encoded bythe nucleic acid sequence of interest. Identifying the intracellularlocation of the mutant GFP thus identifies the intracellular location ofthe protein of interest. The use of such fusion proteins yieldsinformation on the normal cellular role of the protein encoded by thegene of interest, and provides direct measurement of the redox status(and in some embodiments, pH) of the intracellular environment to whichit was targeted. Such an application using wtGFP is described in moredetail, for example, in an article by Olson et al. (J. Cell Biol.,130:639-650,1995).

Also provided are rosGFPs that are sensitive to pH, including forinstance rosGFP2, 4, and 6. Dual sensor GFPs such as these can be usedin any situation in which it is beneficial to monitor or measure both pHand redox status in an environment.

VI. Kits

Kits are provided that contain at least one rosGFP protein, or a nucleicacid molecule e.g., a vector) that encodes such a protein, or both, inone or more contains. The provided kits may also include writteninstructions. The instructions can provide calibration curves or chartsto compare with the determined (e.g., experimentally measured) values.Included are kits that can be used for diagnosis or prognosis of adisease or other condition associated with a change in the redox statusof cell or sub-cellular compartment.

The invention is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Selection and Design of Redox-Sensitive GFPs

In order to create redox-sensitive GFPs, two cysteine residues wereintroduced into GFP that would be within disulfide bonding distance ofeach other, near the chromophore, and on the surface of the protein. Thecysteine residues bring about fluorescence changes based upon whetherthey are reduced or oxidized. In addition, by being on the surface ofthe protein, the cysteines are solvent accessible, which enables them toreversibly respond to redox changes in the surrounding environment.

After close examination of the crystal structure of the S65T variant ofGFP, two borderline suitable sites for the introduction of a pair ofcysteines were chosen. The first site chosen was positions 147 and 204,while the second site chosen was positions 149 and 202. All four aminoacid side-chains at these positions pointed away from the protein'sinterior. The distance between the C_(α)-C_(α) and C_(β)-C_(β) positionsfor site one were 4.6 Å each, and for site two the distance betweenthese positions were 4.8 and 5.9 Å, respectively. These distances didnot agree with previous reports on ideal geometry for the introductionof disulfide bridges in proteins. In addition, neither of these sitepairs were chosen by a disulfide bridge modeling program (Sowdhamini etal., Protein Eng. 3:95-103, 1989). There were, however, some indicationsthat one or both of these pairs of cysteines might be able to form adisulfide bond. Such evidence came from the irregular “bulging” natureof the β-strand encompassing positions 147 and 149, which has previouslybeen shown to move in response to substitution at position 148 (Wachteret al., Structure 6:1267-1277, 1998). Flexibility has been suggested tohelp ensure that a protein can both adjust to the perturbation due toreplacements with cysteine residues as well as to allow the disulfidebridge to assume near-optimal geometry (Matsumura et al., Proc. Natl.Acad. Sci. USA 86:6562-6566, 1989). The 147/204 and 149/202 sites bestfit the criteria.

Example 2 Construction and Expression of Redox Sensitive GFPs

Wild-type GFP contains two cysteine residues at positions 48 and 70. Toavoid possible thiol/disulfide interchange reactions with the newlyengineered cysteines, cysteine 48 and cysteine 70 were replaced withserine and alanine, respectively. Although the substitution C48S did notalter the properties of GFP, C70A appeared to be deleterious toobtaining soluble, fluorescent protein. Since position 70 is locatedwithin the interior of GFP and is in close proximity to the chromophore,it was deemed a mutation-sensitive position and was therefore left as acysteine.

The C48S mutation was introduced into a histidine-tagged version of theS65T variant of GFP in the plasmid pRSETB. This construct served as thetemplate for introduction of the cysteines at sites one (S147C/Q204C)and two (N149C/S202C). All mutations were introduced via the QUIKCHANGE™Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif.), followingthe manufacturer's protocol. Mutations were verified by DNA sequencingof the entire GFP coding sequence.

Mutant protein was recombinantly expressed in Escherichia coli, strainJM109(DE3). Transformed bacteria were grown in four liters of S-LBHmedia at 37° C., stirred at 450 rpm, with 5 liters per minute air flow,and in the presence of 0.27 mM ampicillin. After the culture reached adensity of approximately OD₅₉₅ equal to 0.8, then protein expression wasinduced by addition of isopropyl-β-D thiogalactopyranoside (IPTG) to afinal concentration of 1 mM. At the same time the temperature of theculture was reduced to 16° C. and the culture allowed to grow for anaddition 16 hours. Cells were then harvested by centrifugation at 4° C.in a Beckman KA-9.1000 rotor at 11,800×g for 10 minutes.

The bacterial cell pellet was resuspended in 100 mL of 50 mM HEPES (pH7.9), 300 mM NaCl, 10% glycerol, and 0.1 mM phenylmethylsulfonylfluoride (PMSF). The resuspended cells were sonicated for a total offive minutes, and the lysate clarified twice by centrifugation at35,000×g in a Beckman JA-20 rotor at 4° C. for 20 minutes.

The resultant supernatant was loaded onto a pre-equilibratednickel-nitrilotriacetic acid (Ni-NTA) metal-affinity column (Qiagen,Hilden, Germany). The equilibration and subsequent washing of the columnwas performed with washing buffer (50 mM HEPES (pH 7.9) and 300 mMNaCl). Proteins were eluted from the column by a step gradient of (1)washing buffer plus 20 mM imidazole to remove mostly unwanted proteins,and then (2) washing buffer plus 100 mM imidazole to elute the mutantGFP. To remove the amino-terminal histidine tag and as a furtherpurification step the eluted protein was incubated with 1/50 W/Wγ-chymotrypsin at 22° C. for 22 hours. The protein preparation wasfinally buffer exchanged on a Sephadex® G-25 column. Characteristicyields of mutant GFP protein were in the range of 15 to 100 milligramsand with a purity greater than 95%.

Example 3 Disulfide-Bond Formation and Redox Sensitivity of rosGFPs

Samples of rosGFP2 and GFP S65T (control) were treated with 1 mM DTT or1 μM CuCl₂ and incubated at room temperature for 3-4 hours, then 2 mMN-ethyl maleimide was added to prevent disulfide exchange reactions.Molecular weights were determined by comparison to BENCHMARK proteinladder (InVitrogen, Carlsbad, Calif.). The gel was visualized withCoomassie blue stain.

Results

To verify that the introduced cysteines formed disulfide bonds and toshow that the disulfide bonds were intramolecular, SDS-PAGE was run onredox-sensitive GFP #2 (rosGFP2), harboring the mutationsC48S/S65T/S147C/Q204C, and on a control GFP (C48S/S65T) under reducingand non-reducing conditions. If an intramolecular disulfide bond forms,the resulting polypeptide would be expected to migrate further based onits slightly more compact structure in the denatured form. However, ifan intermolecular disulfide bridge forms, then a large molecular weightband (˜60 kDa) corresponding to a disulfide-linked dimer, would beexpected.

The results (FIG. 4) indicate that the introduced cysteines formintramolecular disulfide cross-links, but are unable to producedisulfide-linked dimers. This can be seen by comparing lanes 11 and 12with lanes 14-16 or lanes 21 and 22 with lanes 17-19 (FIG. 4).

Example 4 Determination of Redox Potentials

Apparent redox potential values for the rosGFPs were found by exploitingthe fact that the fluorescence of the rosGFP chromophores is stronglydependent upon the redox state of the introduced cysteines. Thereforethe redox equilibrium of the rosGFPs with dithiothreitol (DTT_(red)) andoxidized dithiothreitol (DTT_(ox)) was analyzed. The equilibrium for theoxidation of reduced rosGFP by DTT_(red) and its equilibrium constant(K_(eq)) are given by equations 1 and 2.rosGFP_(red)+DTT_(ox)

rosGFP_(ox)+DTT_(red)  (1)K _(eq)=[rosGFP_(ox)] [DTT_(red)]/[rosGFP_(red)] [DTT_(ox)]  (2)When rosGFPs were incubated in the presence of varying concentrations ofDTT_(red) and DTT_(ox) (total DTT_(red)+DTT_(ox)=1 mM), the fractionalamount of reduced rosGFP at equilibrium (R) could be measured over thewhole range from the oxidized to the reduced protein using thechromophore dluorescence (FIG. 20). Based on the SDS-PAGE result thatindicated rosGFPs only form intramolecular disulfide bonds, R can berelated to K_(eq) by equation 3 (Hawkins et al, Biochem J 1991275(2):341-348).R=([DTT_(red)]/[DTT_(ox)])/(K _(eq)+[DTT_(red)]/[DTT_(ox)])  (3)For experimental determination of the equilibrium constants of theGFP:dithiothreitol system, the equilibrium concentrations of DTT_(red)and DTT_(ox) were calculated according to equations 4-6, where[DTT_(red o)] and [DTT_(ox o)] are the initial concentrations ofDTT_(red) and DTT_(ox), respectively, R is the fractional amount ofreduced rosGFP at equilibrium, [rosGFP_(o)] is the initial concentrationof oxidized rosGFP, F is a fluorescence intensity ratio of band Bexcitation (490 nm) versus the isosbestic point (425 nm), and F_(ox) andF_(red) are the 490:425 nm ratios of the completely oxidized and reducedprotein, respectively. By plotting R against the[DTT_(red)]=[DTT_(red o)]−R[rosGFP_(o)]  (4)[DTT_(ox)]=[DTT_(ox o)]+R[rosGFP_(o)]  (5)R=(F−F _(ox))/(F _(red) −F _(ox))  (6)[DTT_(red)]/[DTT_(ox)] ratio and fiting the data to a titration curveaccording to equation 3 (FIG. 21), the K_(eq) for therosGFP1:dithiothreitol system was found to be 2.05×10⁻². The redoxpotential of rosGFP2 at pH 7 and 30° C. (E_(o)′_(rosGFP2)) was thencalculated from the Nernst equation (7), where E_(o)′ is thebiochemist's standard potential of the DTT/DTT_(ox) couple(E_(o DTT)=−0.323 V, at pH 7 and 30° C./ Szajewski and Whitesides 1980), R is the gas constant (8.315 J K⁻¹ mol⁻¹), T is the absolutetemperature (303.15 K), n is the number of transferred electrons (2),and F is the Faraday constant (9.649×10⁴ C mol⁻¹) and found to be −0.272V.E _(o)′_(rosGFP2) =E _(o)′_(DTT)−(RT/nF)×ln K _(eq)  (7)Redox potentials involving the liberation of H⁺ ions are intrinsicallybased on pH. The pH-dependence on the redox potential is more apparentwhen examining the two half-reactions involving rosGFP and DTT (equation8 and 9).rosGFP_(ox)+2H⁺+2e⁻

rosGFP_(red)  (8)DTT_(red)

DTT_(ox)+2H⁺+2e⁻  (9)At equilibrium the concentrations of rosGFP_(ox) and rosGFP_(red) areequal and thus the K_(eq) is equal to the [H⁺]². The stadard redoxpotential of rosGFP2 (E_(o rosGFP2)) at pH 0 could then be determinedfrom equation 10.E _(o rosGFP2) =E _(o)′_(rosGFP2)−(RT/nF)×ln K _(eq)  (10)E_(o rosGFP2) was calculated to be 0.149 V. Equation 11 simplifies theexpression for the pH-dependence of redox potentials involving twoprotons.E _(o) ^(pH) =E _(o)′−60.2 mV×(pH−7)  (11)The pH-dependence on the redox potential therefore changes 60.2 mV witheach pH unit. Experimentally E_(o rosGFP2) varied 65.5 mV per pH unitfrom pH 6 to 8 (correlation coefficient: 0.9999). Therefore, although alinear correlation between pH and E_(o rosGFP2) is observed, thepH-dependence of the rosGFP2 standard potential does not directlycorrespond to this model. The deviation from this model for thepH-dependence of E_(o) may be due in part to the contributions ofcharged residues near the introduced disulfide of rosGFP2 as well aspotentially different pK_(a) values for the cysteine residues(Wunderlich and Glockshuber, Protein Sci 2(5):717-726, 1993; Wunderlichand Glockshuber, J Biol Chem 268(33):24547-24550, 1993)

Example 5 Spectral Analysis of rosGFPs

Spectroscopy and pH Titrations

Absorbance measurements were performed on a Shimadzu 2101spectrophotometer. The molar extinction coefficient of GFP S65T(λ_(280 nm)=19,890 M⁻¹ cm⁻¹) was calculated from its amino acid sequenceas previously described (Gill and von Hippel, Anal. Biochem.182:319-326, 1989), and used to determine protein concentrations of therosGFPs. pH titrations were performed using approximately 200 μg mL⁻¹mutant GFP in 75 mM buffer, 140 mM NaCl and either 1 μM CuCl₂ or 5 mMDTT. According to the desired pH, an appropriate buffer was chosen fromMES, HEPES, or CHES and the final pH was adjusted by addition of HCl orNaOH. The absorbance was then scanned between 250 and 550 nm and theoptical density of the long-wave band B was plotted as a function of pHand fitted to a titration curve to obtain pK_(a) values.

Fluorescence excitation spectra at various pHs were attained on aHitachi F4500 fluorescence spectrophotometer or a Perkin Elmer LS 55luminescence spectrometer at protein concentrations of approximately 100μg mL⁻¹ in the same buffers used for absorbance measurements. Apparentchromophore pK_(a) values were determined by plotting the emissionintensity when excited at band B as a function of pH and fitting thedata to a titration curve (KALEIDAGRAPH™ scientific graphing software).In all cases, the pK_(a) values determined by absorbance andfluorescence differed by no more than +/−0.05 of a pH unit. All plotsand curve fits were made with KALEIDAGRAPH™ scientific graphing software(Abelbeck Software).

Redox Titrations

Fluorescence measurements were performed at 30° C. using a themostatedcuvette holder. Samples consisted of 1 μM GFP in 75 mM HEPES (pH 7.0),140 mM NaCl, 1 mM EDTA, and 1 mM total DTT (mixture of oxidized andreduced forms). To exclude air oxidation, the solutions were degassedand subsequently flushed with nitrogen. In general equilibration wasreached within one hour at pH 7.0. Equilibration of rosGFPs was ensuredby incubating the samples at 30° C. for four hours. The reactionappeared to be at equilibrium, since the ratio of oxidized and reducedprotein, as determined by fluorescence, did not change between 4 and 16hour incubation times.

Spectral Characteristics

To test whether the introduced cysteines affect the spectral propertiesof GFP, absorbance and fluorescence scans were collected at varyingredox potentials. To establish various redox potentials, GFP wasequilibrated with varying concentrations of dithiothreitol (DTT) andoxidized DTT (DTT_(ox)). FIG. 5 shows the absorbance and fluorescenceexcitation spectra of rosGFP2 as a function of redox potential. With adisulfide bond formed under oxidizing conditions, the 400 nm peak (bandA) was maximized whereas the 490 nm peak (band B) was minimized.Conversely, in the absence of a disulfide cross-link achieved withreducing conditions, band B was at a maximum, while band A was at aminimum. The two peaks were separated by a clean isosbestic point at 425nm, indicative of two species interconverting. The redox potential ofrosGFP2 was then determined from the equilibrium constant obtained byplotting the fraction of reduced protein versus the ratio of DTT_(red)to DT_(ox) (see Example 4) and discovered to be −0.272 V.

Introducing a pair of cysteines at the alternative site also yielded amutant protein whose fluorescent properties varied in response to redoxpotential (FIG. 6A). The rosGFP4 variant, having the mutationsC48S/S65T/N149C/S202C, displayed increased band B fluorescence overrosGFP2, however band A fluorescence intensity was almost non-existent.The fluorescence isosbestic point of rosGFP4 was shifted toapproximately 400 nm (FIG. 6B).

Combining the two pairs of cysteine substitutions resulted in a variant(C48S/S65T/S147C/N 149C/S202C/Q204C, rosGFP6), whose properties appearedto be an average of both rosGFP2 and rosGFP4. FIG. 7 shows thefluorescence excitation spectra of rosGFP6. In this variant the dynamicrange of the two excitation peaks as well as the isosbestic point lie inthe middle of the values observed for either rosGFP2 or rosGFP4.

The overall dynamic range of the excitation ratio (δ), determined bydividing the maximum and minimum possible excitation peak ratios, wasfound to be 5.4 for rosGFP6.

Table 5 summarizes the spectroscopic and biochemical parameters of therosGFP variants. TABLE 5 Spectroscopic and Biochemical Properties ofrosGFPs. Name Mutations¹ K_(eq) ² E_(o)′(V)³ δ⁴ rosGFP2 S65T/S147C/Q204C0.0205 −0.272 5.8 rosGFP1 S147C/Q204C 0.0702 −0.288 6.1 rosGFP4S65T/N149C/S202C 0.0561 −0.286 2.6 rosGFP3 N149C/S202C 0.1505 −0.299 4.3rosGFP6 S65T/S147C/N149C/S202C/Q204C 0.0385 −0.280 5.4 rosGFP5S147C/N149C/S202C/Q204C 0.1341 −0.296 7.8¹All variants contain the phenotypically neutral C48S and Q80Rsubstitutions.³Equilibrium constant (K_(eq)) values refer to equilibration of rosGFPswith the DTT_(red)/DTT_(ox) couple.³Redox potentials (E_(o)′) were calculated at pH 7 and 30° C. using theK_(eq) for the GFP-dithiothreitol system (see Appendix).⁴δ is the maximum excitation peak ratio change in number of fold.pH-Sensitivity of rosGFPs

Previous work has shown that, while the fluorescence of wild-type GFP isunaffected throughout the biologically relevant pH range from 6 to 8(Ward and Bokman, Photochem. Photobiol. 35:803-808, 1982), GFP variantsharboring the S65T mutation often exhibit dramatic fluorescence changesover this pH range (Kneen et al., Biophys. J. 74:1591-1599, 1998;Wachter et al., Structure 6:1267-1277, 1998; Elsliger et al.,Biochemistry 38:5296-5301, 1999). To determine if the rosGFPs were alsopH-sensitive, their absorbance and fluorescence were scanned over a widerange of pHs. FIG. 8 shows both absorbance and fluorescence excitationspectra of oxidized rosGFP2 versus pH. The spectra show that, as the pHis increased from 5.2 up to 9.0, the fluorescence intensity of bothexcitation peaks increases. Plotting the intensity at 490 nm as afunction of pH and fitting this to a titration curve with a singleionization constant gave a pK_(a) value of 6.0 (FIG. 9). Titration ofrosGFP2 in the reduced state shifted the pK_(a) to 5.6 (FIG. 10).

Initially it appeared that the pH-sensitivity of the rosGFPs might posea problem for using them as tools to determine in vivo redox potentialsby introducing pH artifacts. Therefore, to produce a pH-insensitiverosGFP, threonine 65 was reverted back to serine, which is the aminoacid found at this position in wild-type GFP.

Not only did the T65S reversion completely eliminate pH-sensitivity overthe range of 6 to 8, but it greatly altered the spectral properties ofthe rosGFPs. FIGS. 11, 12, and 13 show the fluorescence excitationspectra of rosGFPl (C48S/S 147C/Q204C), rosGFP3 (C48S/N149C/S202C), androsGFP5 (C48S/S147C/N149C/S202C/Q204C) at varying redox potentials. Themost striking difference between these rosGFPs and threonine 65containing rosGFPs is the favoring of band A over band B fluorescence.There is also a tendency toward more even excitation of both bands,which is especially evident in the rosGFP3 and rosGFP5 variants.Unexpectedly, the T65S reversion led to a 13-16 mV more reducing redoxpotential (see Table 5).

Subsequent to construction of the variants containing the T65Sreversion, it was determined that, by using a fluorescence excitationratio, any effects induced by pH changes were virtually eliminated. FIG.14 illustrates how the ratio of excitation wavelengths remains aconstant over a large pH range in both the fully reduced or fullyoxidized protein. In other words, the fluorescence increase due toalkalization or fluorescence decrease upon acidification seen in theexcitation spectra of rosGFP2 affects the entire spectrum in the samemanner, and hence the ratio of intensities is unaffected by pH changesin the range of at least pH 6 to 8.5. Therefore, a ratio of theexcitation wavelengths cancels out variations due to pH fluctuations andallows for redox status determination in the absence of pH artifacts.

Since redox reactions involving the liberation of H+ions areintrinsically based on pH (see Example 4), and consequently the pH ofthe cellular compartment of study must be determined, the ability toseparate out the pH and redox sensing capabilities of rosGFP2 wasinvestigated. Excitation of band A was found to result in pH-dependantdual-emission (FIG. 15). In other words, at low pH, blue emissionprevails and at high pH, green emission dominates, with pKaS of 5.6(reduced) to 6.0 (oxidized) dependant upon the redox state of rosGFP2.This phenomenon is a useful means of monitoring pH changes in vivo. Byutilizing a ratio of emission wavelengths, pH variations due to theredox state of the probe are minimized (FIG. 16). Therefore, it ispossible to experimentally separate out the pH and redox contributionsto the fluorescent signal, and thus simultaneous monitoring of redox andpH is possible with the same probe.

Example 6 Structural Basis for Dual-Excitation

The crystal structure of rosGFP2 was solved in order to betterunderstand how disulfide bond formation leads to dual excitation.Crystals belonging to the space group P2₁2₁2 were grown of both theoxidized and reduced forms of rosGFP2 and the crystal structures weresolved by molecular replacement to 1.9 Å and 2.0 Å, respectively. Theprotein crystallized as a dimer, with three molecules in the asymmetricunit. One molecule is related to its dimer mate by a crystallographictwo-fold axis of symmetry, and the other dimer is related to itself by anon-crystallographic two-fold axis of symmetry.

Since the resolution limit of the data collected was fairly high,refinement was performed without imposing any non-crystallographicsymmetry constraints, and thus each of the three molecules in theasymmetric unit were independently refined. Analysis of the differentmolecules in the asymmetric unit after refinement revealed only verysubtle differences, all of which were within experimental error(approximately 0.2 Å) for the atomic positions at this resolution.Interestingly, the surface facing a solvent channel of one of themolecules comprising the non-crystallographic symmetry dimer is verypoorly ordered. This disorder is most certainly reflected in many of therefinement statistics presented in Table 6. TABLE 6 Data Collection andRefinement Statistics for Oxidized and Reduced rosGFP2. Crystal OxidizedReduced Data Collection Total observations 203,865 194,884 Uniquereflections 56,854 38,346 Cell dimensions 186.84, 67.61, 56.08 185.63,67.86, 56.38 (a, b, c; Å) Resolution (Å) 29.7-1.90 28.7-2.00 Highestresolution 1.95-1.90 2.10-2.00 shell (Å) Completeness¹ (%) 99.8 (100) 78.2 (74.3) Multiplicity¹ 3.6 (3.6) 5.1 (5.5) Average I/σ¹ 7.2 (2.3) 8.7(2.2) R_(merge) ^(1,2) 0.058 (0.247) 0.053 (0.304) Refinement SpacegroupP2₁2₁2 P2₁2₁2 Number of molecules³ 3 3 Number of protein 5,220 5,216atoms³ Number of solvent 174 132 atoms³ R_(factor) ⁴ 0.229 0.223 AverageB-factors (Å²) 46.6 49.7 Protein atoms 46.7 49.7 Solvent 44.2 47.4 rmsdeviations Bond lengths (Å) 0.020 0.014 Bond angles (°) 3.111 2.436B-factor correlations (Å²) 7.019 5.779¹Values in parentheses indicate statistics for the highest resolutionshell.²R_(merge) = Σ|I − <I>|/Σ<I>, where I is the observed intensity, and <I>is the average of intensity obtained from multiple observations ofsymmetry related reflections.³Per asymmetric unit.⁴R_(factor) = Σ||F_(o)| − |F_(c)||/Σ|F_(o)|, where F_(o) and F_(c) arethe observed and calculated structure factors, respectively.

The dimer interface is essentially the same as seen for wild-type GFPand the yellow variant of GFP (Yang et al., Nature Biotech. 14:1246-1251, 1996; Wachter et al., Structure 6:1267-1277, 1998). One moleculeof the dimer is tilted approximately 70 degrees with respect to theother molecule based on an imaginary axis drawn from one end of the GFPbarrel structure through the center and out the other end. The dimerinterface is comprised of a small hydrophobic patch consisting ofalanine 206, leucine 221 and phenylalanine 223, a hydrogen bondinteraction between tyrosine 39 and asparagine 149, as well as a numberof hydrophilic contacts involving several bound water molecules. Atpresent it is not known whether the dimer is solution relevant or simplyan artifact created by the crystallization conditions (Palm andWlodawer, Nat. Struct. Biol. 4:361-365, 1997). At any rate, knowledge ofthe dimer interface residues permits future mutational studies aimed atdisrupting dimers without affecting protein stability and folding.

As expected, the introduced cysteines are positioned toward the outsideof the protein and are in excellent arrangement to form a disulfide bondunder oxidizing conditions. They reside along the edge of the dimerinterface and adjacent to bulk solvent. The individual disulfides areseparated by 14 Å from the opposing dimer pair of cysteines andtherefore, as shown by gel electrophoresis, are unable to formintermolecular disulfide bonds and are unlikely to aid in dimerformation. The disulfide bond present in the oxidized structure does nothave ideal geometry, with an average C_(α-C) _(α) distance of 4.0 Å,C_(β)-C_(β) distance of 4.2 Å, S-S distance of 2.0 Å, C_(α)-C_(β)-Sangle of 112°, and C_(β)-S-S angle of 106°. These parameters divergefrom those values seen in other structures of disulfide bond-containingproteins (Sowdhamini et al., Protein Eng. 3:95-103, 1989; Matsumura etal., Proc. Natl. Acad. Sci. USA 86:6562-6566, 1989), and may alsoaccount for or contribute to the observed spectral perturbations.

Example 7 Mammalian Cell Expression and Fluorescence Microscopy

The mutations C48S/T65S/S 147C/Q204C were introduced into the mammalianexpression plasmid pEGFP-N1 (CLONTECH, Palo Alto, Calif.). This plasmidhas the “folding mutation” F64L, which was found not to alter thespectral or redox properties of the rosGFPs. HeLa cells transientlytransfected with this plasmid using Fugene (Boehringer-Mannheim,Germany) were imaged one day post-transfection on a motorized ZeissAxioscope 2 microscope. The temperature of the cells was maintained at37° C. using an open perfusion micro-incubator (Harvard Apparatus Inc.,Holliston, Mass.). Dual-excitation ratio imaging required 400(10) andD480/30x excitation filters, a 505DCXR dichroic mirror, and a D535/40memission filter (Edmund Scientific Company, Omega Optical and ChromaTechnologies, Battleboro, Vt.) alternated by a fast filter changer.Images were collected with a PentaMax cooled CCD camera (PrincetonInstruments). Data was collected and processed using the program Openlab(Improvision, Lexington, Mass.).

In vivo Redox Status

To determine if redox-sensitive GFPs work as indicators of redox statuswithin mammalian cells, rosGFP1 was expressed in the mitochondria ofcultured HeLa cells. FIG. 17 shows the reticular localization pattern ofrosGFP 1 expressed in the mitochondrial matrix via fusion at the DNAlevel to the mitochondrial targeting sequence of the E₁α subunit ofpyruvate dehydrogenase. Upon addition of an oxidizing agent (H₂O₂) thefluorescence excitation ratio (400 nm/490 nm) increased, as expected foran oxidation event (FIG. 18). Conversely, the addition of a reducingagent (DTT) decreased the elevated ratio to a level below the initialratio.

From this experiment several things can be concluded. First, rosGFP1 isable to reversibly respond to induced redox changes within living cells.As seen in FIG. 18, large fluorescence changes accompany the addition ofH₂O₂ or DTT. Second, the results demonstrate that initially rosGFP1 isnot fully reduced or oxidized within mitochondria. Therefore rosGFP1 isable to detect the intrinsic redox potential inside mitochondria,presumably by interacting with endogenous oxidizing and reducing agents.Moreover, the redox potential of rosGFP1 is close to that ofmitochondria and hence rosGFP1 should make an excellent probe forstudying redox changes in mitochondria. Finally, given the initialfluorescence excitation ratio, the percentage of reduced to oxidizedrosGFP1 could be estimated and thus the in vivo redox potential ofmitochondria could be calculated.

Since redox potentials involving the liberation of H+ions areintrinsically based on pH (see Example 4), the GFP S65T/H148D variant(Wachter et al., Structure 6:1267-1277, 1998), with a pK_(a) of 7.8, wasused to examine the pH within the mitochondrion. The inherent pH sensingabilities of rosGFP2 was not utilized in this instance, due to the highpH nature of mitochondria and the high levels of autofluorescence. Thefluorescence excitation ratio of GFP S65T/H148D changed as function ofpH inside the mitochondrial matrix. The addition of oligomycin,carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), orhydrochloric acid resulted in an acidification of the mitochondrialmatrix. On the other hand, addition of NH₄Cl caused the matrix pH toincrease. From these types of additions, the resting pH of themitochondrial matrix of HeLa cells was estimated to be 7.65±0.15 (n=110cells from seven experiments). Due to a slow acidification processduring the experimental setup and data collection process, this valuemay be an underestimate of the true mitochondrial matrix pH. Therefore,the pH value of 7.98±0.07 estimated by Llopis et al. for themitochondrial matrix of HeLa cells was used in subsequent calculations(Llopis et al., Proc. Natl. Acad. Sci. USA 95:6803-6808, 1998).

Based on an average of 23 cells from seven independent experiments, andaccounting for the 60.2 mV per pH unit change in redox potential for areaction involving two protons (see Example 4), the redox potential innormal resting HeLa cell mitochondria was determined to be substantiallymore reducing than −0.3 V.

To investigate the redox potential of the cytosol, rosGFP2 was expressedin the cytosol of HeLa cells. The starting fluorescence amplitude ratioof 400/480 nm excitation was low, as expected for the reducingenvironment of the cytosol of healthy cells. Again, there was a markedincrease and decrease in the ratio upon addition of H₂O₂ and DTT,respectively. However the ratio only recovered to the starting value,indicating that rosGFP2 was fully reduced in the cytosol. Hence theredox potential of the cytosol was estimated to be more reducing than−0.30 V, assuming a pH of 7.34 (Llopis et al., Proc. Natl. Acad. Sci.USA 95:6803-6808, 1998).

Example 8 Factors that Influence the Redox State of rosGFPs In vivo

It is generally thought that the major redox buffer in cells isglutathione and that the ratio of reduced glutathione (GSH) toglutathione disulfide (GSSG) is the crucial parameter for determiningredox status (Meister, Methods Enzymol. 251:3-7, 1995; Deneke, Curr.Top. Cell. Regul. 36:151-180, 2000). On average, the ratio of GSH toGSSG is greater than 100:1 for whole cell determinations on a widevariety of tissue types and total glutathione levels are believed to bearound 1-10 mM (Kosower and Kosower, Int. Rev. Cytol. 54:109-160, 1976;Voet and Voet, Biochemistry, 2nd ed. John Wiley & Sons, Inc., 1995, NewYork, N.Y.; Meister, Methods Enzymol. 251:3-7, 1995). To investigatewhether glutathione may contribute to the redox state of rosGFPs invivo, fluorescence experiments were conducted in vitro.

Incubation of reduced rosGFPl with GSSG resulted in complete oxidationof rosGFP1 in the absence of air oxidation. From this experiment it wasconcluded that rosGFPs expressed in vivo can be oxidized by GSSG.However, since rosGFPs have a lower affinity for electrons than doesglutathione, GSH is unable to reduce oxidized rosGFPs.

Several reports agree that the ratio of GSH to GSSG in mitochondria isbetween 6:1 to 33: 1, with total glutathione approximately 1-2 mM(LêQuôc and LêQuôc, Arch. Biochem. Biophys. 273:466-478, 1989; Bindoliet al., Arch. Biochem. Biophys. 342:22-28, 1997; Lenton et al., Anal.Biochem. 274:125-130, 1999). Substituting these values into the Nernstequation (assuming a mitochondrial pH of 7.98, 37° C., and a standardredox potential of −0.205 V (Szajewski and Whitesides, J. Am. Chem. Soc.102:2011-2026, 1980) for the GSH/GSSG couple) results in a mitochondrialredox potential in the range of −0.210 to −0.230 V. Since the redoxpotential of mitochondria is estimated to be much more reducing than−0.3 V, glutathione alone does not appear to account for such a reducingpotential. Therefore, other redox active agents were investigated.

The NAD⁺/NADH (nicotinamide adenine dinucleotide) pair was considered,because it is present at high concentrations in mitochondria and has avery reducing redox potential. The ratio of NAD⁺ to NADH in the presenceor absence of glucose has been estimated to be anywhere from 1:6 to 10:1in mitochondria (LêQuôc and LêQuôc, Arch. Biochem. Biophys. 273:466-478,1989; Ramirez et al., Biochim. Biophys. Acta 1273:263-267, 1996;Robinson, Methods Enzymol. 264:454-464, 1996). These ratios translateinto redox potentials in the range of −0.328 to −0.382 V at pH 7.98 and37° C. using −0.320 V as the standard potential of the NAD⁺/NADH coupleat pH 7. As a result, this redox couple appeared to be an idealcandidate for maintaining the very reducing environment of themitochondrion. However, redox reactions involving nicotinamides carryout concerted two-electron transfers, whereas thiols undergo twosequential one-electron transfers. As expected, NADH was experimentallyunable to directly reduce rosGFP1 in vitro.

There exists a family of pyridine nucleotide-disulfide oxidoreductases,comprising lipoamide dehydrogenases (LDH), glutathione reductases,thioredoxin reductases, trypanothione reductase, and alkylhydroperoxidereductase. These enzymes all perform homologous reactions ultimatelyinvolving the transfer of NADH or NADPH (nicotinamide adeninedinucleotide phosphate) reducing equivalents to thiols (Carothers etal., Arch. Biochem. Biophys. 268:409-425, 1989). In general terms, thereducing equivalents are transferred from NADH or NADPH through aconcerted two-electron transfer reaction to a bound FAD (flavin adeninedinucleotide) cofactor. FAD then reduces a nearby disulfide bridgethrough two sequential one-electron transfer reactions. In some cases,the electrons are transferred by disulfide exchange to other nearbydisulfides (Ellis and Poole, Biochemistry 36:13349-13356, 1997; Calziand Poole, Biochemistry 36:13357-13364, 1997). Finally, the reduced pairof thiols participates in the reduction of a substrate such aslipoamide, glutathione, thioredoxin, or one of various peroxides. Thekey to how these enzymes carry out their electron transfer reactions islinked to the chemistry of the FAD cofactor, which is able to undergotwo sequential one-electron transfers or a simultaneous two-electrontransfer.

To determine if rosGFPs may be in equilibrium with agents other thanjust glutathione, an in vitro system analogous to a portion of thepyruvate dehydrogenase complex was set up. The system consisted of LDH,lipoate, NADH, and oxidized rosGFP1. The rationale behind thisexperimental setup was to monitor the reduction of rosGFP1 by NADH,through the LDH enzyme and free lipoic acid. FIG. 19 shows that, withall four components of the system present, nearly 75% of rosGFPl isreduced, however removal of LDH, lipoate, or NADH results in less than5% reduction of rosGFPl. Only partial reduction was expected since theredox potential of rosGFP1 (−0.288 V) and the lipoic acid/dihydrolipoicacid couple (−0.29 V; Szajewski and Whitesides, J. Am. Chem. Soc.102:2011-2026, 1980) are very similar. Air oxidized and DTT reducedrosGFP1 were used as standards for obtaining the zero and 100% values.The results of this experiment indicate that it may be possible for NADHreducing equivalents, through various enzymes such as pyruvatedehydrogenase, to be ultimately imposed on rosGFPs in vivo.

This disclosure provides redox-sensitive green fluorescent proteins(rosGFPs), nucleic acids encoding these proteins, and cells transformedwith a nucleic acid encoding a rosGFP. The disclosure further providesmethods of using these molecules to analyze the redox status of, forinstance, a cell or subcellular compartment. It will be apparent thatthe precise details of the methods described may be varied or modifiedwithout departing from the spirit of the described subject matter. Weclaim all such modifications and variations that fall within the scopeand spirit of the claims below.

1. A nucleic acid molecule encoding a mutant green fluorescent protein(GFP) with a fluorescence spectrum that is sensitive to redox status,wherein the mutant GFP shares at least 90% sequence identity with SEQ IDNO: 1 and wherein mutations include: a) a cysteine at one or both of theresidues corresponding to 147 or 149 of SEQ ID NO: 1 and b) a cysteineat one or both of the residues corresponding to 202 or 204 of SEQ IDNO:
 1. 2. The isolated nucleic acid molecule of claim 1, comprising anexpression control sequence.
 3. A recombinant nucleic acid comprisingthe nucleic acid molecule of claim 1 functionally linked to a promoter.4. A host cell comprising a nucleic acid according to claim
 1. 5. Thehost cell of claim 4 wherein the host cell is a bacterial cell, a plantcell, or an animal cell.
 6. The host cell of claim 4 wherein said hostcell is a mammalian cell.
 7. A method of analyzing anoxidation-reduction condition of or in a cell comprising: expressing themutant GFP encoded by the nucleic acid molecule of claim 1 in the cell;and measuring a fluorescence signal from the mutant GFP.
 8. The methodof claim 7, wherein the mutant GFP is expressed as a fusion protein. 9.The method of claim 7, further comprising analyzing a pH condition of orin the cell using the mutant GFP.
 10. The nucleic acid molecule of claim1, comprising mutations selected from the group consisting of: a) theresidues corresponding to 147 and 202 of SEQ ID NO: 1 are cysteine; b)the residues corresponding to 147 and 204 of SEQ ID NO: 1 are cysteine;c) the residues corresponding to 149 and 202 of SEQ ID NO: 1 arecysteine; d) the residues corresponding to 149 and 204 of SEQ ID NO: 1are cysteine; and e) the residues corresponding to 147, 149, 202, and204 of SEQ ID NO: 1 are cysteine.
 11. A method of analyzing anoxidation-reduction condition of or in a cell comprising: expressing themutant GFP encoded by the nucleic acid molecule of claim 10 in the cell;and measuring a fluorescence signal from the mutant GFP.
 12. Theisolated nucleic acid molecule of claim 10, comprising an expressioncontrol sequence.
 13. A recombinant nucleic acid comprising the nucleicacid molecule of claim 10 functionally linked to a promoter.
 14. A hostcell comprising a nucleic acid according to claim
 10. 15. The host cellof claim 14 wherein the host cell is a bacterial cell, a plant cell, oran animal cell.
 16. The host cell of claim 14 wherein said host cell isa mammalian cell.
 17. The nucleic acid molecule of claim 1, comprising anucleic acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO: 10, or SEQ ID NO: 12.